U.S. patent application number 15/071163 was filed with the patent office on 2016-07-21 for use of interleukin 10 mrna transfected macrophages in anti-inflammatory therapies.
This patent application is currently assigned to Ruprecht-Karls-Universitat Heidelberg. The applicant listed for this patent is Ruprecht-Karls-Universitat Heidelberg, Universitat Ulm. Invention is credited to Hugo KATUS, Ziya KAYA, Wolfgang ROTTBAUER, Jan TORZEWSKI, Oliver ZIMMERMANN.
Application Number | 20160206699 15/071163 |
Document ID | / |
Family ID | 44121466 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160206699 |
Kind Code |
A1 |
KAYA; Ziya ; et al. |
July 21, 2016 |
USE OF INTERLEUKIN 10 MRNA TRANSFECTED MACROPHAGES IN
ANTI-INFLAMMATORY THERAPIES
Abstract
The present invention relates to the field of cell-based
therapeutics. Specifically, the invention is concerned with a
composition comprising a macrophage overexpressing interleukin 10
(IL-10) from transfected IL-10 encoding mRNA for use as a
medicament. Moreover, a method for manufacturing a medicament for
treating and/or preventing inflammation or a disease or disorder
associated therewith comprising the steps of obtaining a macrophage
from a sample of said subject, transfecting mRNA encoding IL-10
into said macrophage, and formulating said macrophage in a
composition suitable for administration to the said subject,
whereby the medicament is manufactured. Finally, a kit is provided
for manufacturing such a medicament.
Inventors: |
KAYA; Ziya; (Heidelberg,
DE) ; KATUS; Hugo; (Heidelberg, DE) ;
ZIMMERMANN; Oliver; (Waltenhofen, DE) ; ROTTBAUER;
Wolfgang; (Ulm, DE) ; TORZEWSKI; Jan;
(Immenstadt, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ruprecht-Karls-Universitat Heidelberg
Universitat Ulm |
Heidelberg
Ulm |
|
DE
DE |
|
|
Assignee: |
Ruprecht-Karls-Universitat
Heidelberg
Heidelberg
DE
Universitat Ulm
Ulm
DE
|
Family ID: |
44121466 |
Appl. No.: |
15/071163 |
Filed: |
March 15, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13884888 |
Jul 30, 2013 |
9315558 |
|
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PCT/EP2011/070027 |
Nov 14, 2011 |
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15071163 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/15 20130101;
C12N 5/0645 20130101; C12N 2510/00 20130101; C07K 14/5428 20130101;
A61K 38/2066 20130101; A61P 29/00 20180101; A61K 45/06
20130101 |
International
Class: |
A61K 38/20 20060101
A61K038/20; C12N 5/0786 20060101 C12N005/0786; A61K 45/06 20060101
A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 12, 2010 |
EP |
10 190 950.5 |
Claims
1.-10. (canceled)
11. A pharmaceutical composition comprising a macrophage
overexpressing interleukin 10 (IL-10) from transfected IL-10
encoding mRNA.
12. The pharmaceutical composition of claim 11, wherein the
macrophage further expresses a polypeptide selected from the group
consisting of TGF-.beta., CXCR-4, and CCR2.
13. The pharmaceutical composition of claim 11, wherein the
pharmaceutical composition is useful for treating and/or preventing
inflammation or a disease or disorder associated therewith.
14. The pharmaceutical composition of claim 11, wherein the
macrophage is obtained by a method comprising the steps of: (a)
obtaining a macrophage from a sample of a subject; and (b)
transfecting mRNA encoding IL-10 into the macrophage.
15. The pharmaceutical composition of claim 14, wherein the
pharmaceutical composition is for administration to the subject
from which the macrophage was derived.
16. The pharmaceutical composition of claim 14, wherein the subject
is a human.
17. The pharmaceutical composition of claim 14, wherein the
macrophage is a peritoneal macrophage.
18. The pharmaceutical composition of claim 11, wherein the
macrophage expresses IL-10 at least about 2-fold, at least about
4-fold, at least about 6-fold or at least about 7-fold higher than
a macrophage which has not been transfected by the IL-10 encoding
mRNA.
19. The pharmaceutical composition of claim 11, wherein the
pharmaceutical composition treats or alleviates a disease or
disorder selected from the group consisting of myocarditis,
autoimmune diseases, rheumatoid disease, rheumatoid arthritis, and
multiple sclerosis.
20. The pharmaceutical composition of claim 19, wherein the
myocarditis is autoimmune myocarditis.
21. The pharmaceutical composition of claim 11, wherein the
pharmaceutical composition treats or alleviates local
inflammation.
22. The pharmaceutical composition of claim 21, wherein the local
inflammation is associated with myocarditis, autoimmune disease,
rheumatoid disease, rheumatoid arthritis, or multiple
sclerosis.
23. The pharmaceutical composition of claim 11, wherein the
pharmaceutical composition is formulated for intravenous
administration.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/884,888, filed Jul. 30, 2013, which is the National
Phase of International Patent Application No. PCT/EP2011/070027,
filed Nov. 14, 2011, which claims priority from European Patent
Application No. 10190950.5, filed Nov. 12, 2010. The contents of
these applications are incorporated herein by reference in their
entirety.
[0002] The present invention relates to the field of cell-based
therapeutics. Specifically, the invention is concerned with a
composition comprising a macrophage overexpressing interleukin 10
(IL-10) from transfected IL-10 encoding mRNA for use as a
medicament. Moreover, a method for manufacturing a medicament for
treating and/or preventing inflammation or a disease or disorder
associated therewith comprising the steps of obtaining a macrophage
from a sample of said subject, transfecting mRNA encoding IL-10
into said macrophage, and formulating said macrophage in a
composition suitable for administration to the said subject,
whereby the medicament is manufactured. Finally, a kit is provided
for manufacturing such a medicament.
[0003] Inflammatory reactions represent a challenge for the
treatment of various diseases and disorders with accompanying
inflammation. Such diseases and disorders include e.g. rheumatoid
diseases such as rheumatoid arthritis or autoimmune diseases.
Moreover, cardiac disease such as myocarditis are also accompanied
by inflammatory reactions (Deighton 2009, BMJ 338: b702; Mimori
2008, Nippon Naika Gakki Zasshi 97: 2393-2398).
[0004] Immunosupressive therapies are usually based on steroidal
and non-steroidal antiphlogistics and, in more advanced stages,
chemotherapeutics, antibodies such as Infliximab or, in particular
severe cases, stem cell therapies. The current immunosuppressive
therapies are unspecific and, thus, affect healthy cells and
tissues as well (Senolt 2009, Autoimmun Rev 9: 102-107; Fautrel
2009, Arthritis Rheum 61: 425-434; Snowden 2008, Autoimmunity 41:
625-631; Ho 2009, Aliment Pharmacol Ther 29: 527-534). Adverse side
effects of e.g. steroid-based immunosuppressive therapies are
metabolic syndrome, diabetes or osteoporosis. A local or specific
therapeutic approach may avoid at least some of the aforementioned
drawbacks.
[0005] Myocarditis is determined by various factors and is most
often a cause of cardiac diseases in young adults (Gupta 2008, Nat
Clin Pract Cardiovasc Med 5: 693-706). Although acute myocarditis
is often entirely cured within several weeks, some cases become
chronic and may cause even more severe diseases such as dilatative
cardiomyopathy and chronic heart failure. The latter disorder is
notwithstanding modern therapeutic intensive care associated with a
high risk of mortality and morbidity (Bendayan 2008, J Heart Lung
Transplant 27: 698-699; December 1. 994, N Engl J Med 331: 1564;
Kawai 1999, Circulation 99: 1091-1100; Cihakova 2008, Adv Immunol
99: 96-114; Hunt 2009, Circulation 119: 391-479). Recently,
inflammatory processes and viral infections have been discussed as
a cause of myocarditis (McKenna 1997 Heart 77:549-552; Kuhl 2005,
Circulation 111: 887-893).
[0006] Interleukin 10 (IL-10) has been known for a long time as an
anti-inflammatory cytokine. It represses the antigen presentation
and the T-cell activation. Moreover, IL-10 inhibits the production
of TH1 cytokines and promotes B-cell survival, proliferation and
antibody production. It has been applied already in several
therapeutic approaches (Mosser 2008 Immunol Rev 226: 205-218; Grutz
2005, Journal Leukocyte Biol 5: 3-15; Thummler 2009, Z Rheumatol
68: 337-339; Vandenbark 2008, Immunology 123: 66-78). Moreover,
IL-10 has also been used in gene therapy or cell therapeutic
approaches. However, due to the use of viral vectors those
approaches were less efficient and accompanied by severe side
effects (El-Shemi 2004, Kidney Int 65: 1280-1289; Spight 2005, Am J
Physiol Lung Cell Mol Physiol 288: 251-265; Pinderski 2002, Circ
Res 90: 1064-1071). mRNA transfection of adult progenitor cells has
been reported to be less harmful and applicable for human therapy
(WO 2007/090647).
[0007] Thus, there is a need for an improved cell-based therapy for
inflammatory disease which avoids the aforementioned drawbacks.
[0008] The technical problem underlying the present invention can
be seen as the provision of means and methods for complying with
the aforementioned needs. The technical problem is solved by the
embodiments characterized in the claims and herein below.
[0009] Accordingly, the invention relates to a composition
comprising a macrophage overexpressing interleukin 10 (IL-10) from
transfected IL-10 encoding mRNA for use as a medicament.
[0010] As shown in the following examples, in vitro overexpression
of IL-10 in macrophages resulted in a 7-fold increased IL-10
production. In vivo by trend higher levels of IL-10 and less
inflammation was detected within the myocardium of treated compared
with control mice. Mice treated with IL-10 overexpressing
macrophages presented a significantly better performance in running
wheel tests. Echocardiography revealed a trend towards an improved
cardiac function in treated mice.
[0011] Accordingly, overexpression of IL-10 in macrophages could
reduce inflammation and improve cardiac performance in a murine
model of myocarditis. The use of genetically modified macrophages
can thus facilitate a targeted therapy of local inflammatory
processes. As the nucleofection technique is basically GMP-adapted
an in vivo use in humans seems principally possible. Finally, the
results shown in the following examples strongly suggest that the
therapeutic approach of the present invention can be transferred to
other inflammatory diseases characterized by local inflammation
such as Crohn's disease, vasculitis, rheumatoid arthritis
autoimmune diseases, rheumatoid disease, or multiple sclerosis.
[0012] The term "macrophage" as used herein refers to a white blood
cell which is usually present in various body tissues or body
fluids. They are usually mobile cells which migrate by amoeboid
movements. Macrophages are formed by differentiation of monocytes.
Said monocytes and macrophages are phagocytes which are involved in
innate immunity as well as adaptive immunity of vertebrate animals.
Macrophages have a pivotal function in phagocytosis of cellular
debris and pathogens, and to stimulate lymphocytes and other immune
cells to respond to a pathogen by an inflammatory reaction.
[0013] Macrophages as referred to herein are, preferably,
identified by specific expression of marker proteins. A macrophage
according to the present invention can be identified by at least
one of the following marker proteins: CD14, CD11b, F4/80
(mice)/EMR1 (human), lysozyme M, MAC-1/MAC-3 and CD68. More
preferably, the macrophage according to the present invention can
be identified and/or isolated by CD11b and/or CD68. Viable
macrophages can be isolated from a sample by cell separation
techniques such as flow cytometry, magnetic bead-based affinity
purification techniques or affinity chromatography using antibodies
or aptamers which specifically recognize the aforementioned marker
proteins and, in particular, CD11 b.
[0014] Macrophages can be isolated from various different body
tissues and body fluids of a subject including blood, lymph,
liquor, saliva, and others. More preferably, the macrophage
according to the present invention can be isolated from a body
tissue such as spleen or peritoneal tissue and, most preferably,
from the peritoneal tissues. Preferably, a subject according as
meant in this specification is a mammal. Preferably, said mammal is
a rodent, such as a mouse or a rat, a pet, such as a cat or dog, or
a farming animal such as a horse, pig or cow. Most preferably, the
mammal is, however, a human.
[0015] The term "interleukin 10 (IL-10)" as used herein refers to a
cytokine having strong anti-inflammatory properties. It is also
known as cytokine-synthesis inhibitory factor. IL-10 is secreted by
monocytes and TH2 cells as well as regulatory T-cells. Its strong
anti-inflammatory properties are based on its capability of
eliciting a reduction of T-cell activation as well as a reduction
of antigen presentation. IL-10 protects the organism from excessive
inflammatory reactions. The structure of IL-10 of several species
including rodents such as mice as well as humans is well known in
the art. The human IL-10 polypeptide forms a homodimer. Each
subunit is 178 amino acids long. The gene for IL 1 is located on
chromosome 1 (Eskdale 1997, Immunogenetics 46(2): 120-8; Zdanov
1995, Structure 3(6): 591-601). For example, the Accession number
NM_010548.2 shows the corresponding sequences of murine IL-10,
whereas the Accession number depicted in NM_000572.2 or P22301
shows the corresponding sequences of human IL-10.
[0016] It will be understood that the present invention also
encompasses variants of the aforementioned specific IL-10
polypeptides. Such variants may be chemically modified or
genetically engineered variants having essentially the same
anti-inflammatory properties as the aforementioned IL-10
polypeptides. Moreover, variants may be homologous or orthologous
polypeptides from other species. Preferably, such variants have an
amino acid sequence being at least 40%, at least 50%, at least 60%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 98% or at least 99% identical to the
amino acid sequence of the specif. The term "identical" as used
herein refers to sec aforementioned IL-10 polypeptides. Sequence
identity characterized by determining the number of identical amino
acids between two nucleic acid sequences or amino acid sequences
wherein the sequences are aligned so that the highest order match
is obtained. It can be calculated using published techniques or
methods codified in computer programs such as, for example, BLASTP,
BLASTN or FASTA (Altschul 1990, J Mol Biol 215: 403). The percent
identity values are, preferably, calculated over the entire amino
acid sequence. A series of programs based on a variety of
algorithms is available to the skilled worker for comparing
different sequences. In this context, the algorithms of Needleman
and Wunsch or Smith and Waterman give particularly reliable
results. To carry out the sequence alignments, the program PileUp
(Higgins 1989, CABIOS 5: 151) or the programs Gap and BestFit
(Needleman 1970, J Mol Biol 48: 443; Smith 1981, Adv Appl Math 2:
482), which are part of the GCG software packet (Genetics Computer
Group 1991, 575 Science Drive, Madison, Wis., USA 53711), may be
used. It will be understood that these variants shall also have
essentially the same anti-inflammatory properties as the
aforementioned IL-10 polypeptides.
[0017] The IL-10 polypeptide is transcribed in accordance with the
present invention from an mRNA molecule which has been transfected
into the macrophage. The nucleotide sequence for such an mRNA
molecule or the nucleotide sequence for a DNA molecule encoding it
can be established by the skilled artisan without further ado for a
given polypeptide such as the IL-10. The mRNA to be transfected can
be chemically synthesized or can be obtained from a DNA template by
in vitro transcription. Suitable techniques are well known in the
art and commercial kits for carrying out such in vitro
transcription reactions are available. Nucleotide sequences for
IL-10 encoding mRNA or DNA are well known in the art and are,
preferably, described by such nucleotide sequences can also be
applied for deriving a suitable nucleotide sequence for the IL-10
encoding mRNA to be applied in accordance with the present
invention. Preferably, the mRNA to be transfected into the
macrophage in accordance with the present invention differs from
naturally occurring mRNA in the poly-A-tail and/or the
cap-structure at the 5'end of the mRNA molecule, i.e. it is an
artificial mRNA differing in its composition from the endogenously
transcribed mRNA encoding IL-10. Moreover, nucleotide analogs can
be used for the synthesis of the mRNA. Various nucleotide analogs
have been reported to increase the stability of a mRNA. More
preferably, the poly-A tail and/or cap-structure of the artificial
mRNA which is transfected into the macrophage confers increased
stability to the mRNA molecule. The mRNA to be transfected can be
homologous or heterologous with respect to the macrophage, i.e. the
mRNA and the macrophage may be from the same species or may be from
different species.
[0018] The macrophage to be applied in the composition according to
the present invention shall be transfected with mRNA encoding
IL-10. The mRNA for the IL-10 is, thus, exogenously supplied to the
macrophage rather than being transcribed from its endogenous IL-10
gene. Techniques for introducing mRNA into a cell such as a
macrophage are well known in the art. A cell may be transfected
with mRNA by physical techniques such as micro-injection of the
mRNA or may be transfected by electroporation based methods such as
nucleofection (see Pascal 2005, J Neurosci Methods 142(1): 137-43;
Aluigi 2006, Stem Cells, 24(2): 454-461 Maasho 2004, Journal of
Immunological Methods, 284(1-2): 133-40; WO 2007/090647). For the
nucleofection technique, commercial kits are available (e.g., from
Lonza Cologne GmbH). The transfected mRNA encoding IL-10 will be
translated into the IL-10 polypeptide upon transfection into the
macrophage.
[0019] By "overexpressing" it is meant that the IL-10 polypeptide
is produced in the transfected macrophage compared to a
non-transfected macrophage in a statistically significant higher
amount. Whether an amount of IL-10 produced by the macrophage is
significantly higher can be determined by standard statistically
techniques. Preferably, the transfected macrophage in the
composition to be applied according to the present invention
expresses IL-10 at least 2-fold, at least 4-fold, at least 6-fold
or at least 7-fold higher than a macrophage which has not been
transfected by the IL-10 encoding mRNA. The person skilled in the
art can determine an amount of mRNA which results in such an
overexpression without further ado. Suitable amounts of mRNA are,
preferably, within the range of 3 to 9 .mu.g RNA for between
1.times.10.sup.6 to 5.times.10.sup.6 macrophages.
[0020] Thus, the macrophage referred to in accordance with the
composition of the present invention is, preferably, obtained by a
method comprising the steps of: [0021] (a) obtaining a macrophage
from a sample of a subject; and [0022] (b) transfecting mRNA
encoding IL-10 into said macrophage.
[0023] The macrophage can be obtained from a tissue sample of a
subject, preferably, the subject to be treated by the composition.
The tissue sample is, preferably, a peritoneal sample. The
macrophage is, preferably, obtained from the sample by affinity
purification based on the CD11b marker protein present on the
macrophages, well known in the art. The corresponding Accession
numbers of the murine CD11b and the human CD11b sequences are
shown, e.g., in NM_008401.2 and NM_000632.3 or P11215,
respectively. However, the techniques for isolating macrophages
referred to elsewhere herein can be also applied. The isolated
macrophages are, subsequently, transfected with mRNA encoding IL-10
in an amount resulting in the production of a therapeutically
effective dose of IL-10 upon translation into IL-10 polypeptide.
Details are also described elsewhere herein. The transfected
macrophages can then be formulated in a suitable composition and
applied as a medicament.
[0024] Moreover, it will be understood that the macrophages
according to the present invention are, preferably, cultivated upon
transfection for a time and under conditions sufficient for
allowing effective translation of the mRNA into IL-10 polypeptide
before the composition is formulated. Moreover, it is to be
understood that only viable macrophages should be used for the
composition of the present invention. Accordingly, prior to
formulation as a composition of the present invention, the
macrophages are, preferably, cultivated for at least 3 hours, at
least 4 hours, at least 5 hours or at least 6 hours after
transfection. Moreover, the non-viable macrophages shall be removed
prior to formulation of the composition according to the present
invention. More preferably, the composition is formulated and the
medicament is to be applied between 6 to 72 hours, more preferably,
between 6 and 24 hours and, most preferably, at about 6 hours after
transfection.
[0025] The aforementioned macrophage is applied according to the
present invention in a composition. Such a composition, in
accordance with the present invention, comprises additional
compounds, preferably, a pharmaceutically acceptable carrier. The
pharmaceutically acceptable carrier must be acceptable in the sense
of being compatible with the macrophage and other ingredients of
the composition and being not deleterious to the recipient thereof.
The said carrier may be a gel or a liquid. Examples of liquid
carriers are phosphate buffered saline solution, water,
physiological saline, Ringer's solutions, and the like. Suitable
carriers comprise those mentioned above and others well known in
the art, see, e.g., Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa. Preferably, the pharmaceutically
acceptable carrier is a physiological buffer or medium in which the
macrophage is maintained and/or can be applied to a subject to be
treated in accordance with the invention. Moreover, the composition
may comprise additional drugs which improve or facilitate the
anti-inflammatory effect of the IL-10 overexpressing macrophage.
Preferably, a further drug to be included into the composition
according to the present invention is TGF-.beta. well known in the
art. The TGF-.beta. can be TGF-.beta.1, TGF-.beta.2 or TGF-.beta.3.
The corresponding sequences of murine and human TGF-.beta.1 are
shown, e.g., in Accession number NM_011577.1 and NM_000660.4 or
P01137, respectively. The corresponding sequences of human
TGF-.beta.2 are shown, e.g., in Accession number
NM_0011355599.2
[0026] The composition referred to herein above shall be used as a
medicament. The term "medicament" as used herein refers to a
pharmaceutical composition comprising the macrophage described
above as pharmaceutical active compound. The medicament is,
preferably, administered in a therapeutically effective dose. A
therapeutically effective dose refers to an amount of the
macrophage to be used in the composition applied according to the
invention which prevents, ameliorates or treats the symptoms
accompanying a disease or disorder referred to in this
specification. Therapeutic efficacy and toxicity of a given drug
including the cell based therapeutic according to the present
invention can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., ED50 (the dose
therapeutically effective in 50% of the population) and LD50 (the
dose lethal to 50% of the population). The dose ratio between
therapeutic and toxic effects is the therapeutic index, and it can
be expressed as the ratio, LD50/ED50.
[0027] The dosage regimen will be determined by the attending
physician and other clinical factors. As is well known in the
medical arts, dosages for any one patient depends upon many
factors, including the patient's size, body surface area, age, the
particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. Dosage recommendations shall be indicated in the
prescribers or users instructions in order to anticipate dose
adjustments depending on the considered recipient.
[0028] The medicament in accordance with the present invention is,
preferably, suitable for topical as well as systemic applications.
It can be administered at least once in order to treat or
ameliorate or prevent a disease or condition recited in this
specification. However, the medicament can also be administered
more than one time.
[0029] The medicament referred to in this specification may be used
for human or non-human therapy of various diseases or disorders.
Preferably, said medicament is used for treating and/or preventing
inflammation or a disease or disorder associated therewith. More
preferably, the medicament is used for the treatment and/or
prevention of a disease or disorder selected from the group
consisting of: myocarditis, autoimmune diseases, rheumatoid
disease, rheumatoid arthritis, and multiple sclerosis. Most
preferably, the medicament is for the treatment and/or prevention
of myocarditis.
[0030] The symptoms accompanying the aforementioned diseases and
disorders are well known to those skilled in the art and are
described in standard text books of medicine.
[0031] Advantageously, it has been found in the studies underlying
the present invention that macrophages which are transfected with
mRNA encoding the anti-inflammatory cytokine IL-10 can be used for
treating and/or preventing inflammatory diseases or disorders such
as myocarditis. Specifically, it was found that murine macrophages
from peritoneal tissue which have been transfected with an
effective amount of IL-10 encoding mRNA efficiently inflammatory
processes in a mouse myocarditis model (see Examples below).
Adverse side effects which had been reported previously for
macrophages transfected with DNA plasmids or viral vectors did not
occur. Moreover, the transfection of mRNA is a highly efficient
process which basically complies with the GMP provisions for human
application.
[0032] In addition, the inventors were able to demonstrate
beneficial effects after injection of IL-10 overexpressing
macrophages both in a prophylactic and therapeutic approach: In the
first clinical setting of macrophage application, autoimmune
myocarditis was induced by subcutaneous injection of troponin I.
IL-10 overexpressing macrophages were injected on day 14, 21 and 28
when autoimmune myocarditis was completely developed. This setting
represents a therapeutic approach as the genetically modified
macrophages were injected after myocarditis became clinically
apparent. In the second clinical setting, IL-10 overexpressing
macrophages were injected simultaneously with troponin I
immunization on day 0, 7 and 14. Here a prophylactic approach is
displayed, that should prevent development of autoimmune
myocarditis before clinical symptoms became evident.
[0033] The present invention, in principle, contemplates, thus, a
method for treating and/or preventing inflammation or a disease or
disorder associated therewith in a subject suffering therefrom
comprising the steps of: [0034] (a) obtaining a macrophage from
said subject; [0035] (b) transfecting mRNA encoding IL-10 into said
macrophage; [0036] (c) formulating said macrophage in a composition
suitable for administration to the said subject; and [0037] (d)
administering said composition to the subject in a therapeutically
effective dosage.
[0038] In a preferred embodiment of the composition for use as a
medicament according to the present invention, said composition is
for application in the subject from which the macrophage was
derived. Accordingly, an autologous cell therapy is envisaged, i.e.
the donor subject and the acceptor subject of the macrophage is
identical.
[0039] In a preferred embodiment of the composition for use as a
medicament according to the present invention, said macrophage
further expresses a polypeptide which facilitates the
anti-inflammatory properties of the macrophage comprised by the
composition of the invention. Preferably, said polypeptide is
TGF-.beta..
[0040] In a preferred embodiment of the composition for use as a
medicament according to the present invention, said macrophage
comprised by the composition of the invention further expresses a
polypeptide which facilitates migration of the macrophage to a
target tissue in an organism. Preferably, said polypeptide is a
chemokine-receptor and, more preferably, CXCR-4 and/or CCR-2. The
corresponding murine CXCR-4 sequences are shown, e.g., in Accession
number NM_009911.3. The corresponding human CXCR-4 sequences are
shown, e.g., in Accession number NM_001008540.1 or P61073.1. The
corresponding sequences of murine CCR-2 are shown, e.g., in
NM_009915.2. The corresponding sequences of human CCR-2 are shown,
e.g., in NM_001123041.2 or P41597.1. These chemokine-receptors are
particularly useful for attracting the macrophage to the
myocarditis in the heart.
[0041] The present invention also relates to a method for
manufacturing a medicament for treating and/or preventing
inflammation or a disease or disorder associated therewith
comprising the steps of: [0042] (a) obtaining a macrophage from a
sample of said subject; [0043] (b) transfecting mRNA encoding IL-10
into said macrophage; and [0044] (c) formulating said macrophage in
a composition suitable for administration to the said subject,
whereby the medicament is manufactured.
[0045] Details on how to obtain and transfect the macrophage and on
how to formulate the macrophage as a composition for the use as a
medicament can be found elsewhere in this specification. It will be
understood that the aforementioned method is carried out according
to GMP standards.
[0046] In a preferred embodiment of the method of the invention,
said medicament is for the treatment and/or prevention of a disease
or disorder selected from the group consisting of: myocarditis,
autoimmune diseases, rheumatoid disease, rheumatoid arthritis, and
multiple sclerosis.
[0047] In another preferred embodiment of the method of the
invention, said macrophage expresses IL-10 at least 2-fold, at
least 4-fold, at least 6-fold or at least 7-fold higher than a
macrophage which has not been transfected by the IL-10 encoding
mRNA.
[0048] Finally, the present invention also contemplates a kit for
manufacturing a medicament for treating and/or preventing
inflammation or a disease or disorder associated therewith
comprising [0049] (a) a device for isolating macrophages from a
sample of a subject; [0050] (b) a transfection reagent for mRNA;
and [0051] (c) (i) mRNA encoding IL-10 or (ii) a polynucleotide
encoding IL-10 together with reagents for transcription
thereof.
[0052] The term "kit" as used herein refers to a collection of the
aforementioned components, preferably, provided in separately or
within a single container. The container also comprises
instructions for carrying out the method of the present invention.
These instructions may be in the form of a manual or may be
provided by on a data storage medium or device such as an optical
storage medium (e.g., a Compact Disc).
[0053] Devices and reagents referred to in accordance with the kit
of the invention have been further specified elsewhere herein and
are also well known to the person skilled in the art.
[0054] All references cited in this specification are herewith
incorporated by reference with respect to their entire disclosure
content and the disclosure content specifically mentioned in this
specification.
FIGURES
[0055] FIGS. 1A-1B: mRNA-nucleofection of different macrophage
populations
[0056] Two different populations of macrophages were compared for
overexpression of EGFP (Enhanced Green Fluorescent Protein): lienal
vs. peritoneal macrophages. Respectively, 3 .mu.g mRNA were
transfected and EGFP expression was measured 24 h later by FACS
analysis. MOCK transfected cells and EGFP nucleofected cells were
compared. Top row: peritoneal macrophages; bottom row: lienal
macrophages. EGFP expression was significantly higher and more
reproducible in peritoneal than in lienal macrophages. In this
representative experiment, 40% EGFP positive lienal macrophages and
80% EGFP positive peritoneal macrophages were detected.
[0057] FIG. 2: Interleukin-10 ELISA--time kinetics
[0058] IL-10 levels were analyzed in vitro in the supernatant of
macrophages 3, 6, 24, 30, 48 and 72 h after nucleofection of 3
.mu.g mRNA for IL-10. MOCK transfected macrophages were used as a
negative control. 6 h after mRNA-nucleofection, the maximum IL-10
concentration could be detected. Per 10.sup.5 macrophages in the
positive group, 399.+-.59 pg/ml IL-10 and in the negative group,
57.+-.6 pg/ml IL-10 could be detected. In the negative control,
MOCK transfection resulted in a marginal increase of IL-10 levels
right after the nucleofection procedure. Within 24 h, IL-10 levels
decreased to baseline values again. Mean values of three
independent experiments are depicted.
[0059] FIG. 3: Interleukin-10 ELISA--concentrations
[0060] In vitro the IL-10 concentration in the supernatant of
macrophages was analyzed 6, 24 and 48 h after nucleofection of 3, 6
and 9 .mu.g mRNA. A significant increase in IL-10 production could
be detected when nucleofection was performed with raising amounts
of mRNA. This observation could be confirmed at all time points.
Mean values of four independent experiments are depicted.
[0061] FIGS. 4A-4H: Morphological tracking of injected macrophages
by Red Fluorescent Protein (RFP)
[0062] In one group, myocarditis was induced by immunization with
troponin I as described earlier (Goser 2006, Circulation 114:
1693-1702). RFP.sup.+ macrophages overexpressing IL-10 were
injected intraveneously. Healthy A/J wildtype mice, who did not
receive any macrophages served as a negative control. One week
after injection, the recipient mice were sacrificed and heart,
lung, kidney, liver, spleen, skin and lymph nodes were analyzed for
RFP.sup.+ macrophages to detect distribution of the cells within
the host body. No RFP-positive macrophages could be detected within
the lungs, kidney, skin, liver and muscle. In the spleen and lymph
nodes a weak red background staining--similar to RFP--could be
detected, but no differences were seen between the positive and
control mice. Significantly more RFP.sup.+ macrophages could be
detected within the heart compared with the control mouse. These
findings may indicate a targeted therapy to sites of
inflammation.
[0063] FIG. 5: Functional tracking of injected macrophages by IL-10
production
[0064] Top: In healthy A/J WT mice, i.e. mice without induction of
myocarditis, IL-10 overexpressing macrophages were administered.
MOCK transfected macrophages represented the negative control.
After 24 h, the spleen was extracted and homogenized. The IL-10
concentration was measured in the supernatant of the splenocytes by
ELISA. The inventors could detect a basal IL-10 production in
splenocytes of WT A/J mice after administration of MOCK transfected
macrophages, but significantly more IL-10 was produced in
splenocytes after IL-10 overexpressing macrophages were injected.
Again this could indicate a targeted therapy as macrophages seem to
home back to organs of the mononuclear phagocyte system when
inflammation is missing.
[0065] Bottom: IL-10 levels were determined in the blood serum of
the above MOCK and IL-10 nucleofected A/J WT mice. 10.sup.6
macrophages were transfected respectively and 24 h later IL-10
levels were determined in the serum by ELISA. Neither in MOCK nor
in IL-10 transfected mice relevant IL-10 concentrations were
detectable. Once more this finding could indicate a targeted
therapy with no/low systemic side effects.
[0066] FIG. 6: Histological analysis of myocardial inflammation
[0067] Determination of the Histo-Score: myocardial sections were
paraffin embedded and inflammation was quantified by H&E
staining. A graduation of infiltrating cells was made 20%-wise
(grade 1 to 5). After induction of myocarditis IL-10 overexpressing
macrophages were injected on day 0, 7 and 14. On day 21 animals
were sacrificed and histologigal analysis was performed. MOCK
transfected macrophages represented the control group. Overall
myocardial inflammation was significantly decreased after treatment
with IL-10 overexpressing macrophages compared with controls. In
this representative experiment, the mean Histo-Score was 2.2.+-.0.6
for IL-10 treated mice vs. 3.5.+-.0.5 for the MOCK control
(p=0.024).
[0068] FIGS. 7A-7B: Immunohistochemical staining in myocardial
inflammation
[0069] A) A detailed subanalysis of inflammatory cells was
performed in myocardial sections. CD3.sup.+ lymphocytes, CD68.sup.+
macrophages and IL-10 was analyzed after treatment with IL-10 and
MOCK transfected macrophages. By trend more IL-10, less lymphocytes
and less macrophages were detected in IL-10 treated mice compared
with MOCK transfected animals. B) Additionally, overall
infiltrating cells were analyzed by H&E staining which were
reduced after IL-10 treatment (see also FIG. 6). Myocardial
fibrosis was detected by Masson's-Trichrom staining. Again, a
semiquantitative score (grade 1 to 5) was applied. No significant
differences could be detected between both groups which could be a
result of the early analysis right 21 days after the first
induction of myocardial inflammation. Thus, it might be too early
to detect relevant differences for myocardial fibrosis.
Representative experiments are depicted.
[0070] FIG. 8: Antibody titers
[0071] An enzyme-linked immunosorbent assay (ELISA) was established
to measure the titer of autoantibodies against cTnI. Mice treated
with intravenous application of IL-10 overexpressing macrophages
showed significant lower titers of autoantibodies compared to mice
treated with MOCK transfected macrophages; see Example 1.7.
[0072] FIG. 9: Functional analysis of heart failure by
echocardiography
[0073] Echocardiography was performed on day 21 after induction of
myocarditis in IL-10 and MOCK treated mice. The ejection fraction
(EF) and the fractional shortening (FS) were not significantly
different between both groups, but by trend a better left
ventricular function was detected for IL-10 treated heart failure
mice (p=0.156 respectively).
[0074] FIG. 10: Physical performance in the running wheel
[0075] Mice were treated with IL-10 overexpressing macrophages and
compared with a MOCK transfected control group as described above.
Between day 14 and 21, i.e. within the third week, the inventors
analyzed the walking time and distance the mice performed
voluntarily in the running wheel. The inventors could detect a
better physical performance as walking distance and time spent in
the wheel were significantly higher in IL-10 treated animals
compared with the MOCK group. Total exercise time (p=0.055),
average exercise time per day (p=0.043), total walking distance
(p=0.046) and average walking distance per day (p=0.020) were
compared with MOCK transfected control mice.
EXAMPLES
[0076] The invention will be merely illustrated by the following
Examples. The said Examples shall, whatsoever, not be construed in
a manner limiting the scope of the invention.
Example 1
Materials and Methods
[0077] 1.1 Isolation, Characterization and Culture of Murine
Macrophages
[0078] A/J mice (Jackson Lab; USA) older than 8 weeks served as
donors for macrophage isolation. After cervical dislocation, 15 ml
of a Hanks Balanced Salt Solution (HBSS; Gibco, USA) were injected
intraperitoneally. HBSS contained 10% HBSS, 1% HEPES and 2% FCS
(fetal calf serum). After 5 minutes of incubation, the buffer was
aspirated with a syringe and the cell suspension was centrifugated
at 300 g. Then the pellet was resuspended in 450 .mu.l PBS with
0.3% BSA (bovine serum albumine) and 2 mM EDTA. 50 .mu.l of
magnetically labeled human anti-mouse CD11b antibody (Miltenyi
Biotec, Germany) was added for 20 minutes. CD11b positive
monocytes/macrophages were extracted using a MS column (Miltenyi
Biotec, Germany) according to manufacturers' instructions. Per
mouse about 0.5.times.10.sup.6 macrophages could be isolated.
Macrophages of two mice were pooled to obtain one million cells for
subsequent mRNA-nucleofection. Additionally murine macrophages were
isolated from the spleen. The spleen was extracted, cut into small
pieces and homogenized by squeezing the cells through a 30 .mu.m
pre-seperation-filter (Miltenyi Biotec, Germany). Afterwards, CD11b
positive monocytes/macrophages were purified as described
above.
[0079] 1.2. Preparation of RFP.sup.+ Macrophages
[0080] For in vivo tracking of administered macrophages, cells were
isolated from RFP.sup.+-transgenic mice. All cells of these mice
express the Red Fluorescent Protein (RFP) which makes donor
macrophages visible in the host. RFP.sup.+-transgenic reporter mice
were kindly provided by Prof H. J. Fehling (Institute for
Immunology, Ulm University, Luche 2007 Eur J Immunol 37: 43-53). To
avoid cell rejection, the RFP.sup.+ C57BL/6-inbred reporter mice
were backcrossed with A/J mice. To confirm and accelerate the
backcross procedure, a genome scanning (Jackson Lab, USA) was
performed to identify the mice with the most autologous genotype.
Thus, a specific backcross to the A/J background was possible.
[0081] 1.3 Vector Construction and In Vitro Transcription
[0082] The murine 537 bp IL-10 gene (Accession Number NM_010548)
was cloned into the pBluescript II SK(+) vector and purchased from
Eurofins (France). For the experiments, the IL-10 plasmid was
diluted 1:10 and cloned into a pcDNA3.1N5-His TOPO vector
(Invitrogen, USA). The plasmid was enzymatically linearized using
ScaI and in vitro transcription was performed as described earlier
(Wiehe 2006 Regen Med 1: 223-234; Wiehe 2007 J Cell Mol Med 11:
521-530).
[0083] 1.4 mRNA-Nucleofection of IL-10 and EGFP
[0084] 10.sup.6 macrophages were dissolved in 100 .mu.l mouse
macrophage Nucleofector.RTM. kit (AMAXA, Germany) and 3 .mu.g of
IL-10 mRNA were added. For nucleofection, program X-01 of the
Nucleofector.RTM. device (AMAXA, Germany) was used. Afterwards
macrophages were cultured in RPMI medium (PAA, Austria) containing
10% FCS and 1% PSG (penicillin-streptomycin-glutamine).
[0085] Alternatively, 3 .mu.g of mRNA for Enhanced Green
Fluorescent Protein (EGFP) were nucleofected. The DNA plasmid that
served as a template for in vitro transcription was kindly provided
by Dr. Peter Ponsaerts (University of Antwerp, Belgium). EGFP
positive cells were detected by fluorescence activated cell sorting
using a FACSDiva (Becton, Dickinson and Company, USA) as described
in detail in Wiehe et al. (Wiehe 2007 J Cell Mol Med 11:
521-530).
[0086] 1.5 Interleukin-10 ELISA
[0087] a) Time-Dependent Effects of IL-10 Overexpression In
Vitro
[0088] 10.sup.6 macrophages were nucleofected with 3 .mu.g mRNA for
IL-10 as described above. Cells were seeded in 48 well plates and
cultured for 3, 6, 24, 30, 48 and 72 h in 500 .mu.l RPMI, 10% FCS
and 1% PSG medium per well. The IL-10 concentration was determined
in the supernatant using a DuoSet ELISA Development System (R&D
Systems, USA). For detection, a Blue Star HRP Substrate (Diarect
AG, Germany) was utilized. The ELISA was performed according to
manufacturers' instructions.
[0089] b) Dose-Dependent Effects of IL-10 Overexpression In
Vitro
[0090] 3, 6 and 9 .mu.g of mRNA for IL-10 were used for
nucleofection. The IL-10 concentration in the supernatant was
analyzed 6, 24 and 48 h later.
[0091] c) Determination of IL-10 Serum Level In Vivo
[0092] IL-10 levels were determined in the blood serum of healthy
A/J mice after injection of MOCK and IL-10 nucleofected
macrophages. No inflammation was induced in these mice. 10.sup.6
macrophages were transfected respectively and 24 h later IL-10
levels were determined in the serum by ELISA.
[0093] 1.6 Murine Myocarditis Model
[0094] a) Production and Purification of Cardiac Troponin I
(cTnI)
[0095] Murine cTnI (Accession Number shown in P48787) was
transformed into E. coli bacteria. The protein was expressed and
purified by anion-cation-exchange as well as affinity
chromatography. The quality of the extract was analyzed by SDS gel
and Western Blot. A detailed protocol was provided earlier (Goser
2006, Circulation 114: 1693-1702; Goser 2005, Circulation 112:
3400-3407; Kaya 2002, J Immunol 168: 1552-1556).
[0096] b) Immunization
[0097] This procedure was described in detail earlier (Goser 2006,
Circulation 114: 1693-1702; Goser 2005, Circulation 112: 3400-3407;
Kaya 2002, J Immunol 168: 1552-1556). In brief, for induction of
myocardial inflammation cTnI and complete Freud's adjuvant (CFA)
were mixed in a 1:1 ratio. Afterwards an emulsion was generated.
Female A/J mice at the age of 6 weeks were subcutaneously injected
with 120 .mu.g of the emulsion on day 0 and 7. A control group was
immunized with an emulsion of the pre- and post-fraction from the
cTnI and CFA alone.
[0098] c) In Vivo Application of Modified Macrophages
[0099] Effects of timing: i) IL-10 overexpressing macrophages were
injected on day 0, 7 and 14 into the tail vein of A/J recipient
mice. To confirm a sufficient mRNA-nucleofection, IL-10 levels were
determined in vitro in the supernatant of macrophages as described
above. This represents a prophylactic approach as therapy is
initiated simultaneously with triggering of myocardial
inflammation. Macrophages that underwent the nucleofection
procedure without addition of IL-10 mRNA served as controls (i.e.
MOCK transfection). ii) additionally macrophages were administered
after myocarditis was clinically apparent on day 14, 21 and 28.
This represents a therapeutic approach.
[0100] Effects of the inflammatory setting: IL-10 overexpressing
macrophages were intraveneously injected into A/J mice with and
without induction of myocarditis to analyze cell distribution.
[0101] 1.7 Antibody Titers
[0102] An enzyme-linked immunosorbent assay (ELISA) was established
to measure the titer of autoantibodies against cTnI. The 96-well
plates were coated overnight at 48.degree. C. with 5 mg/mL cTnI
(100 mL/well) dissolved in bicarbonate buffer (0.1 M NaHCO.sub.3/34
mM Na.sub.2CO.sub.3, pH 9.5). 1.times.PBS/0.05% Tween 20 served as
washing buffer. Plates were then coated with 1% Gelatine (Cold
Water Fish, Sigma, 300 mL/well). After an incubation period of 2 h
at 37.degree. C. and rinsing, IgG (Sigma A2554) diluted to 1:5000
was applied for detection (1 h at room temperature, 100 mg/well).
Dilution series of serum samples were performed as follows: 1:100,
1:400, 1:1600, 1:6400, and 1:25 600. Blue Star HRP substrate
solution (Diarect) was then applied for 30 min at room temperature
(100 mL/well) and the colour reaction was stopped with 0.3 M
H.sub.2SO.sub.4. All samples were measured in duplicate. Optical
densities of each well were determined using a microplate reader
set to 450 nm. The antibody endpoint titer of each mouse was
determined as the highest positive dilution of antibody; see FIG. 8
for result.
[0103] 1.8 Functional Analysis of Heart Failure
[0104] a) Transthoracic Echocardiography
[0105] Echocardiography was performed using an ATL-HDI 9000
(Philips, The Netherlands) device with a 10 MHz linear transducer.
In the longitudinal axis enddiastolic and endsystolic diameter,
ejection fraction (EF), fractional shortening (FS) and heart rate
were determined. Mice were investigated after anaesthesia with 1-2%
(v/v) isoflurane.
[0106] b) Treadmill Exercise Test
[0107] A running wheel was placed in each cage and every mouse had
its own device. The individual voluntary walking distance and time
was measured. After one week of adaption the parameters obtained in
the third week after application of IL-10 overexpressing
macrophages were analyzed.
[0108] 1.9 Histological Analysis
[0109] a) Determination of the Histo-Score
[0110] Serial cross-sections of 5 mm thickness each through the
entire heart were prepared and stained with haematoxylin and eosin
(H&E) in order to define the level of inflammation. Masson's
tri-chrome staining was used to determine the extent of collagen
deposition. An independent examiner blinded to the treatment arm of
the respective specimens explored every fifth cross-section and
classified them according to the six-tier scoring system published
previously (Kaya 2002, J Immunol 168: 1552-1556; Goser 2006,
Circulation 114:1693-702; Kaya 2008, Circulation 118: 2063-72).
[0111] b) Determination of Myocardial Fibrosis
[0112] Myocardial fibrosis was semi-quantitatively analyzed by
Masson's-Trichrom staining according to the pattern above (Kaya
2002, J Immunol 168: 1552-1556; Goser 2006, Circulation 114:
1693-702; Kaya 2008, Circulation 118: 2063-72).
[0113] c) Immunohistochemical Analysis of Myocardial
Inflammation
[0114] Myocardial inflammation was assessed by immunohistochemistry
as described earlier (Zimmermann 2005, Int J Cardiol 104: 92-100).
In brief, CD3 positive lymphocytes, CD68 positive macrophages and
IL-10 were detected within the myocardium by the
avidin-biotin-peroxidase complex. Myocardial sections were fixed
with formalin and incubated with 0.1% proteinase (Sigma, USA) for
10 minutes. A 1% H.sub.2O.sub.2/methanol solution was used to block
endosomal peroxidase activity. Unspecific antigens were blocked
with rabbit or goat serum in a 1:10 dilution. A polyclonal rat
anti-mouse CD68 antibody (dilution 1:50; clone FA-11, Acris
Antibody GmbH, Germany), a polyclonal goat anti-mouse CD3 antibody
(dilution 1:50; Santa Cruz, USA) and a polyclonal goat anti-mouse
IL-10 M-18 antibody (dilution 1:100; Santa Cruz, USA) were used as
a primary antibody. A biotinylated rabbit anti-goat (Dako, Germany)
and goat anti-rat antibody (BioLegend, USA) was diluted 1:200 and
served as a secondary antibody, respectively. Myocardial sections
were analyzed every 25 .mu.m for each antigen using an Axioskop 2
plus microscope (Zeiss, Germany). Thus, at least 20 sections were
available which represent about 400 high power fields (i.e. 40-fold
magnification). A semiquantitative score system was applied for
IL-10. Staining intensity was indicated from 0 (i.e. no IL-10
detection) to "+++" (i.e. strong IL-10 detection). CD3 and CD68
positive cells were counted under the light microscope
(cells/mm.sup.2). Immunohistochemical analysis was performed
blinded by experienced investigators.
[0115] 1.10 Statistical Analysis
[0116] Data were analyzed with the Kruskal-Wallis test followed by
the Mann-Whitney U test to explore the significance between
treatment groups. P<0.05 was considered significant. The
statistical software SPSS (version 15.0) was used for all
calculations.
Example 2
Results
[0117] 2.1 mRNA-Nucleofection of Different Macrophage
Populations
[0118] To establish an optimized protocol for mRNA-nucleofection,
two different populations of macrophages were compared for
overexpression of EGFP: lienal vs. peritoneal macrophages.
Respectively, 3 .mu.g mRNA were transfected and EGFP expression was
measured 24 h later by FACS analysis. A significantly higher
expression of EGFP could be observed in peritoneal than in lienal
macrophages (FIG. 1). On average, 20% (1-40%) EGFP positive lienal
macrophages and 82% (80-85%) EGFP positive peritoneal macrophages
could be detected. Furthermore, isolation of macrophages from the
abdominal cavity is faster, easier and more reproducible compared
with the isolation of lienal macrophages. Thus, peritoneal
macrophages were isolated for this study.
[0119] 2.2 Interleukin-10 ELISA
[0120] a) Time-Dependent Effects of IL-10 Overexpression In
Vitro
[0121] After nucleofection of macrophages with 3 .mu.g mRNA for
IL-10, IL-10 levels in the supernatant were analyzed 3, 6, 24, 30,
48 and 72 h later. MOCK transfected macrophages were used as a
negative control. 6 h after mRNA-nucleofection, the maximum IL-10
concentration could be detected which represents a 7-fold increase
compared with the negative control. Per 10.sup.5 macrophages in the
positive group, 399.+-.59 pg/ml IL-10 and in the negative group,
57.+-.6 pg/ml IL-10 could be detected. As mRNA-nucleofection
represents a transient form of genetic cell modification IL-10
level constantly decreased within a couple of days. In the negative
control, MOCK transfection resulted in a marginal increase of IL-10
levels right after the nucleofection procedure. Within 24 h IL-10
levels decreased to baseline values again. Additionally, the
supernatant of the macrophages has been removed regularly to
analyze different intervals of IL-10 production. Transfected
macrophages produced nearly all IL-10 within the first 24 hours
(data not shown). FIG. 2 shows the mean IL-10 concentrations (pg/ml
per 10.sup.5 macrophages) of three independent experiments.
[0122] b) Dose-Dependent Effects of IL-10 Overexpression In
Vitro
[0123] 3, 6 and 9 .mu.g of mRNA for IL-10 were used for
nucleofection. The IL-10 concentration in the supernatant was
analyzed 6, 24 and 48 h later (pg/ml per 10.sup.5 macrophages). A
significant increase of IL-10 could be observed when nucleofection
was performed with rising amounts of mRNA.
[0124] This observation was confirmed at all time points
investigated. Also the above described time kinetics could be
confirmed in this experiment. Median values from four independent
experiments are depicted in FIG. 3.
[0125] 2.3 Targeted Therapy Using IL-10 Overexpressing
Macrophages
[0126] a) Tracking of Macrophages by Detection of RFP
[0127] RFP.sup.+ C57BL/6-inbred reporter mice were backcrossed with
A/J mice. From these mice, RFP.sup.+ macrophages were isolated. The
injected macrophages could be tracked in WT mice by fluorescence
microscopy and thus distinguished from innate macrophages. No
relevant amounts of macrophages could be detected within the liver,
skin, muscle, kidney, lungs, spleen and the lymph nodes.
Significantly more RFP.sup.+ macrophages could be detected in the
heart compared to the non-treated WT control mouse. These findings
probably indicate a targeted therapy to sites of inflammation (FIG.
4).
[0128] b) Tracking of Macrophages by IL-10 Production
[0129] IL-10 overexpressing macrophages were injected intravenously
into healthy A/J WT mice without induction of myocarditis. 24 h
later the spleen was extracted, homogenized and IL-10 levels were
determined by ELISA in the supernatant of the splenocytes. MOCK
transfected macrophages served as a negative control. A
significantly higher IL-10 production could be detected in the
treatment group compared with the negative control. The mean IL-10
concentration was 350 vs. 420 pg/ml (p<0.05; FIG. 5, top). This
result could indicate, that in healthy animals without any
inflammation, IL-10 overexpressing macrophages leave the
vasculature soon to be stored in the spleen. This represents a
physiological pattern as macrophages usually home in organs of the
macrophage monocytic phagocytic system (MMPS) if there is no active
inflammation. Quiescent macrophages can be recruited from these
tissues in the case of active inflammation.
[0130] c) Determination of IL-10 Serum Levels In Vivo
[0131] IL-10 levels were determined in the blood serum of MOCK and
IL-10 nucleofected healthy WT A/J mice. 10.sup.6 macrophages were
transfected respectively and 24 h later IL-10 levels were
determined in the serum by ELISA. Neither in MOCK nor in IL-10
transfected mice a relevant IL-10 concentration was detectable; see
FIG. 5, bottom. This observation indicates that there is no
significant systemic increase in IL-10 levels and thus no relevant
adverse side effects are expected.
[0132] 2.4 Histological Analysis
[0133] a) Determination of the Histo-Score
[0134] IL-10 was overexpressed in macrophages by nucleofection of 6
.mu.g mRNA. 10.sup.6 macrophages were injected intraveneously into
A/J WT mice after induction of myocarditis. 21 days after first
immunization the mice were sacrificed and H&E staining of
myocardial sections was performed. MOCK transfected mice
represented the negative control. Myocardial inflammation was
analyzed by application of the Histo-Score. A significant reduction
of infiltrating cells could be seen within the myocardium after
application of IL-10 overexpressing macrophages (FIGS. 6 and 7A).
The Histo-Score was 2.9.+-.0.6 for IL-10 treated mice vs.
4.6.+-.0.4 for the control group (p=0.004; FIG. 6).
[0135] b) Determination of Myocardial Fibrosis
[0136] Myocardial fibrosis was detected by Masson's-Trichrom
staining. A semiquantitative score (grade 1 to 5) was applied in
analogy to the Histo-Score. No significant differences could be
detected between both groups which could be a result of the early
analysis right 21 days after the first induction of myocardial
inflammation. Thus, it might be too early to detect relevant
differences for myocardial fibrosis. Representative experiments are
depicted (FIG. 7B).
[0137] 2.5 Antibody Titers
[0138] Three weeks after first immunization with cardiac troponin
antibody titers against cardiac troponin was measured. Mice treated
with intravenous application of IL-10 overexpressing macrophages
showed significant lower titers of autoantibodies compared to mice
treated with MOCK transfected macrophages (p<0.05; FIG. 8).
[0139] 2.6 Functional Analysis of Heart Failure
[0140] a) Transthoracic Echocardiography
[0141] Three weeks after first immunization with cardiac troponin,
echocardiograpy was performed. MOCK transfected macrophages were
injected as a control. By trend IL-10 treatment resulted in a
better ejection fraction and fractional shortening than application
of MOCK transfected macrophages. The fractional shortening (FS) was
38.8.+-.1.4% in IL-10 treated mice and 34.6.+-.2.0% in MOCK
transfected animals (p=0.156). The ejection fraction (EF) was
77.+-.2 in the IL-10 treated mice and 71.+-.3 in the control group
(p=0.156). A statistical significance was missed. FIG. 9 depicts a
representative experiment. Each group consisted of 10 mice
respectively.
[0142] b) Treadmill Exercise Test
[0143] Mice were treated as described above. During the third week
after first immunization with troponin, voluntary physical
performance was investigated using a running wheel. The time and
distance was measured that mice acquired in the third week after
intraveneously injection of the genetically modified cells.
Spontaneous physical activity was significantly increased after
injection of IL-10 overexpressing macrophages. The mean walking
distance per day was 5.6.+-.0.7 km in the treatment group vs.
3.8.+-.0.6 km in the control group (p=0.020; FIG. 10). Also total
exercise time (p=0.055), mean exercise time per day (p=0.043) and
total walking distance (p=0.046) were compared with MOCK
transfected control mice.
[0144] 2.7 Dose-Dependent Effects of IL-10 Therapy
[0145] The above experiments were carried out again, but this time
only 5.times.10.sup.5 genetically modified macrophages were
injected intraveneously. Now the inventors were not able to detect
any significant differences for echocardiography and treadmill
exercise tests between IL-10 overexpressing macrophages and the
MOCK control. Also histological analysis could not detect any
significant differences for myocardial CD3, CD68 and IL-10
distribution. Thus, a dose dependent efficacy of the therapeutic
principle presented herein seems possible (data not shown).
Example 3
Discussion
[0146] In the present study, the inventors investigated an in vivo
application of IL-10 overexpressing macrophages for
anti-inflammatory therapy in a mouse model of autoimmune
myocarditis. The inventors could demonstrate that myocardial
inflammation could be reduced and clinical performance was
increased after injection of genetically modified macrophages.
Furthermore, the inventors gained evidence that their therapeutic
approach displayed only a local action that was focused within the
myocardium. There was no systemic impact of this therapy as the
inventors could not detect elevated IL-10 plasma level after
intravenous injection of IL-10 overexpressing macrophages.
Significantly more RFP.sup.+ positive macrophages could be detected
within the myocardium of TnI immunized mice in contrast to other
tissues. Nearly no macrophages were seen in the liver, skin,
muscle, lungs and the kidneys whereas an unspecific red background
staining could be detected in the spleen and in lymph nodes. The
inventors postulate that macrophages rather selectively migrate
into the inflamed tissue, release IL-10 locally and thus mediate
their anti-inflammatory effects specifically. Thus, in a clinical
setting, adverse side effects could be reduced by this targeted
therapy.
[0147] Finally, an in vivo application in patients with acute
myocarditis seems basically possible as the nucleofection technique
is adapted to the GMP-guidelines and the isolation of the
macrophages can be arranged in an autologous setting (Wiehe 2006
Regen Med 1: 223-234; Wiehe 2007 J Cell Mol Med 11: 521-530). This
fact is one major advantage of the therapy of the invention as
earlier approaches were limited by the use of viral vectors, low
efficacy and a potential procedural toxicity (El-Shemi 2004, Kidney
Int 65: 1280-1289; Pinderski 2002, Circ Res 90: 1064-1071; Spight
2005, Am Physiol Lung Cell Mol Physiol 288: 251-265). As the
inventors present a universal approach for a targeted
anti-inflammatory therapy this method could be transferred to other
processes characterized by local inflammation as Crohn's disease,
vasculitis or rheumatoid arthritis.
[0148] The inventors applied two clinical settings of macrophage
application: i) autoimmune myocarditis was induced by subcutaneous
injection of troponin I. IL-10 overexpressing macrophages were
injected on day 14, 21 and 28 when autoimmune myocarditis was
completely developed. This setting represents a therapeutic
approach as the genetically modified macrophages were injected
after myocarditis became clinically apparent. ii) IL-10
overexpressing macrophages were injected simultaneously with
troponin I immunization on day 0, 7 and 14. Here a prophylactic
approach is displayed, that should prevent development of
autoimmune myocarditis before clinical symptoms became evident. In
both settings, the inventors were able to demonstrate beneficial
effects after injection of IL-10 overexpressing macrophages.
[0149] One challenge of the present study is the histological
analysis of myocardial sections. Myocardial inflammation appears in
clusters and so it is difficult to get an exact number of
macrophages and lymphocytes as many cells may be located in the
same area and cannot be distinguished separately. The inventors
tried to solve this methodological limitation by analysis of at
least 400 high power fields and calculation of the mean cell number
per mm.sup.2 for each antigen per heart.
[0150] Within recent years there were numerous attempts to extent
the standard heart failure therapy in myocarditis. Unspecific
immunosuppressive therapy using prednisone, cyclosporine or
azathioprine was introduced without convincing results (Mason 1995,
N Engl J Med 333: 269-275). In viral dilated cardiomyopathy, a
specific antiviral therapy with interferon .beta.-1b was
investigated. In a phase I study some beneficial effects could be
detected for this therapy (Kuhl 2003, Circulation 107: 2793-2798)
but a phase II study and another clinical trial could not confirm
the initial promising results (Schultheiss 2008, Circulation 118:
2312 (Abstract); Zimmermann 2010, J Card Fail 16: 348-356). Against
this background, new anti-inflammatory therapies based on IL-10
overexpression seem worth to be further followed.
[0151] The anti-inflammatory power of the Th2-associated cytokine
IL-10 has been utilized in a couple of recent studies. IL-10
mediates its immunomodulatory properties by inhibition of Th1
cells, macrophages and cytokines like NF-.kappa.B, TNF-.alpha.,
IL-1 or IL-6 and thus protects the organism from overwhelming
proinflammatory conditions (Matsumori 2001, Heart Fail Rev 6:
129-136). Recently, IL-10 was suggested as the effective part of
some anti-inflammatory therapies as fenofibrate (Maruyama 2002, J
Atheroscler Thromb 9: 87-92), quercetin--a flavonoid--(Milenkovic
2010, J Pharm Pharm Sci 13: 311-319), mesenchymal stem cells (Weil
2010, Surgery 148: 444-452), immunoglobulins (Matsumori 2010, Circ
Res 106: 1533-1540) and methotrexate (Zhang 2009, Mediators Inflamm
2009: 389720). In contrast to these animal studies, patients with
myocarditis showed a worse outcome when IL-10 serum levels were
elevated (Fuse 2005, Eur J Heart Fail 7: 109-112; Nishii 2004, J Am
Coll Cardiol 44: 1292-1297). This observation may be explained by
the fact, that in fulminant myocarditis more pro-inflammatory
cytokines are produced and consequently more IL-10 is released for
counterbalancing. Nishio and colleagues administered recombinant
IL-10 subcutaneously in a murine model of autoimmune myocarditis
caused by the encephalomyocarditis virus (Nishio 1999, Circulation
100: 1102-1108). They could report a significantly higher survival
rate in the treatment group compared with the control group.
Furthermore myocardial lesions were smaller and the levels of
TNF-.alpha., IL-2 and iNOS in the heart were lower in IL-10 treated
mice. Interestingly, the beneficial effects were only seen when
IL-10 was begun on the day of virus inoculation (i.e. prophylaxis)
whereas no effects were seen when IL-10 was administered later
(i.e. therapy) (Nishio 1999, Circulation 100: 1102-1108). This
observation could be confirmed in a murine model of fatal group B
streptococcus sepsis (Nishio 1999, Circulation 100: 1102-1108). In
a rat model of myocarditis, IL-10 was also protective when a
plasmid vector expressing the IL-10 cDNA was transferred into the
tibialis anterior muscle by electroporation (Palaniyandi 2004, Eur
J Immunol 34: 3508-3515; Watanabe 2001, Circulation 104:
1098-1100). In contrast, in the present non-viral murine
myocarditis model the inventors could detect beneficial effects of
an IL-10 therapy in a prophylactic and in a therapeutic setting
respectively.
[0152] In this study, the inventors report for the first time that
application of IL-10 overexpressing macrophages could display
beneficial effects in a murine model of autoimmune myocarditis.
Recent attempts to introduce IL-10 therapy to a clinical setting
were limited by the use of viral vectors, low efficacy and a
potential procedural toxicity (El-Shemi 2004, Kidney Int 65:
1280-1289; Pinderski 2002, Circ Res 90: 1064-1071; Spight 2005, Am
Physiol Lung Cell Mol Physiol 288: 251-265). The therapeutic
approach of the present invention is adapted to the GMP-guidelines
(Wiehe 2006, Regen Med 1: 223-234; Wiehe 2007, J Cell Mol Med 11:
521-530) and based on an autologous background. Thus, basically a
bridge to a clinical application can be built. As nucleofection
results in a transient overexpression of IL-10 there is no
permanent genetic modification of the macrophages which furthermore
supports a clinical use (Wiehe 2006, Regen Med 1: 223-234; Wiehe
2007, J Cell Mol Med 11: 521-530). Modifications of the mRNA could
potentially influence the duration of IL-10 expression. Finally,
the inventors present a first proof of principle for a new
therapeutic approach in autoimmune myocarditis.
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